Adenovirus vectors are among the most popular vehicles for gene therapy protocols currently used to attack cancers, because of their use of genetic manipulation, their ability to be prepared at high titres and their ability to efficiently transfer genetic material into appropriate target cells. However, adenovirus gene therapy vectors have several targeting disadvantages; (i) they infect non-tumor cells that express the Coxsakie and Adenovirus Receptor, CAR, thus diverting vectors from the target cell and (ii) infect target tumor cells via cell-specific targeting mechanisms based either on a biochemical manipulation or genetic alterations of the virus. We developed two adenoviruses, Ad-D2R and Ad-HSV1-tk, that delivery PET reporter genes, whose expression can be monitored with positron- labeled probes and demonstrated their efficacy in transferring these reporter genes to murine liver. We will now use these PET reporter viruses and PET reporter probes to investigate the efficacy of bi-specific antibodies (Bi-Ab) and molecular conjugates to redirect adenovirus gene therapy vectors to tumor cells. Specifically, we will investigate the ability of (i) a Bi-Ab directed to the therapy vectors to tumor cells. Specifically, we will investigate the ability of (i) a Bi-Ab directed to the [epidermal growth factor (EGF) receptor][adenovirus knob protein] and (ii) a [soluble CAR][EGF] complex to redirect Ad-D2R and Ad-HSV 1-tk to xenografts of A431 epidermoid carcinoma cells (which express 2-3x106 EGF receptors) xenografted to nude mice subcutaneously, in the lung, in the liver and in the bone marrow. In collaboration with David Curiel at the U. of Alabama we will identify, from phage display libraries, peptides and single chain antibody fragments that react from EGFR, incorporate these peptides into both the fiber protein HI loop and the hexon hypervariable 5 region of HSV-1 tk expressing adenoviruses, and ablate the CAR binding site. Targeting of these viruses for A431 xenografts will be examined by microPET. Only by using PET receptor gene/reporter probe technology can we obtain the resolution, quantitation and non- invasive characteristics necessary to monitor the distribution and gene transfer capability of these biochemically and genetically retargeted adenovirus gene therapy vectors.